去泛素化酶CYLD缺失导致小鼠焦虑样行为的机理研究

CYLD, a deubiquitinating enzyme, plays important and abundance roles in many biological processes. It is a critical regulator of diverse cellular processes, ranging from proliferation and differentiation to inflammatory responses. Recent studies demonstrate that CYLD regulates synaptic function in nervous system. It is reported that there are higher expressions in the nucleus accumbens, the amygdala, the hypothalamus, the prefrontal cortex and the hippocampus, which have typically been considered as the brain regions of the anxiety system. There is the highest expression in the striatum, which is a major entry structure of neural circuit between the cerebral cortex and the basal ganglia. Recently, we observed an anxiety-like behavior (behavior in the open arm of an Elevated Plus Maze and the open field) in Cyld knock-out mice. The expression levels of both the GABAA and GABAB receptors, number of microglia marked by Iba-1 are increased in striatum of Cyld knock-out mice. Whether do they cause CYLD deficiency-induced anxiety-like behavior? Whether is the striatum included in the anxiety circuitry? How do they regulate the activities of anxiety circuitry? The physiological functions of CYLD in the anxiety-like behavior remains unexplored. Therefore, we utilize Cyld knock-out mice and wild-type mice with litter-mate comparisons to explore the mechanism of GABA signaling mediated by CYLD deficiency-induced anxiety-like behavior, at the whole animal, tissue, cellular and molecular levels. 1. The expression levels of Cyld gene and CYLD protein will be selectively down-regulated or up-regulated within striatum using shRNA. The relationship between CYLD and the anxiety-like behavior will be identified using behavior tests in those mice and Cyld knock-out mice. 2. In vivo double-channel extracellular recordings, in vivo multi-channel extracellular recordings, optogenetics and slice patch-clamp technique will be employed to examine the changes in synaptic activities, neuronal circuitry, oscillation, pharmacological effects of endogenous and exogenous agonist and antagonist GABAA and GABAB receptors. 3. Western blotting, immunohistochemical and biochemistrical methods will be employed to examine the roles of GABAergic system and neuroinflammation in CYLD-mediated anxiety-like behavior. The accomplishment of this project will gain insight into the function of CYLD in the anxiety. It may have implications for the targets of novel anxiolytic treatments.

虽然去泛素化酶CYLD在免疫系统起重要作用，近年发现CYLD也参与神经系统功能的调节，并且在介导焦虑的脑区高表达，在纹状体中的表达量最高。我们已发表和预实验结果发现敲除CYLD的小鼠出现焦虑样行为，其纹状体神经元的突触传递、GABAA和GABAB受体及小胶质细胞活化等发生改变。纹状体的GABA能系统和炎性异常是否CYLD缺失导致焦虑的起因？仅在纹状体改变CYLD表达是否改变小鼠的焦虑行为？本立项将针对上述科学问题结合行为、在体多通道、脑片膜片钳、光遗传、免疫组化等方法，围绕以下几方面开展研究：CYLD缺失小鼠纹状体分子及环路变化与焦虑样行为的关系、以及下调纹状体内CYLD是否导致小鼠焦虑样行为；纹状体GABA能系统异常和神经炎症变化是否CYLD敲除导致焦虑样行为的机理，揭示GABA能系统与炎症变化的因果关系。通过阐述纹状体CYLD异常导致焦虑行为的机制，为寻找治疗焦虑的靶点提供借鉴和参考。

CYLD是一种关键的去泛素化酶，在多个脑区高表达，它在神经系统中的作用正受到关注。我们发现敲除CYLD的小鼠表现焦虑样行为，但仍不清楚：CYLD是如何参与焦虑样行为的调控？仅改变纹状体中CYLD的表达是否改变小鼠的焦虑样行为？表达CYLD量最高的纹状体中的中型多棘神经元（MSN）、小清蛋白中间神经元（PV-IN）和神经胶质细胞各起的作用？为回答上述科学问题，本项目以Cyld基因敲除小鼠、纹状体小清蛋白中间神经元特异性敲除Cyld的PV-Cre x Cyldflox/flox（PV-Cyld KO）小鼠及特异性敲除纹状体D1-MSN/D2-MSN的Cyld 基因小鼠Drd1-Cyldflox/flox-Cre (Drd1, Cyld KO)和Drd2-Cyldflox/flox-Cre (Drd2, Cyld KO)为实验材料，通过行为学、脑片全细胞膜片钳记录、场电位记录、在体细胞外技术记录、蛋白免疫印迹、免疫共沉淀、免疫组化等实验发现：敲除Cyld基因导致小鼠产生焦虑样行为，但未造成小鼠基本运动能力损伤和抑郁；神经炎症是影响小鼠焦虑行为的关键因素；与Cyld+/+小鼠相比，Cyld-/-小鼠的功率频谱密度在δ频段的power值显著减小，而θ频段, β频段, γlow，γhigh频段的节律无明显差异。CYLD通过调控-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体（AMPAR）的关键亚基谷氨酸受体1（GluA1）和谷氨酸受体2（GluA2）的表达与功能，调节纹状体MSN和PV-INs的形态、放电活性和突触功能，CYLD与 GluA1和GluA2相互作用，调控GluA1及GluA2 K63链接的泛素化修饰，维持AMPAR的稳定性。CYLD介导的GluA1和GluA2的去泛素化是控制AMPARs功能的关键调节信号，对突触的组织、功能和可塑性至关重要，将有助于对CYLD在焦虑样行为和调节情绪记忆中作用的理解。